BIOCHEMISTRY #12 BY: AMMAR AL-HABAHBEH فيصل الخطيب. October 11, 2012

Transcription

1 BIOCHEMISTRY #12 د. فيصل الخطيب October 11, 2012 BY: AMMAR AL-HABAHBEH

2 The Beginning Degradation and synthesis does not occur in a single step but in several steps where sequence of steps converts starting material into end product, this called metabolic pathway it can be anabolic or catabolic, all pathways together makes metabolism. The first example of metabolic pathway is glycolysis where glucose is converted to 2 molecule of Pyruvate in 10 steps. Glycolysis is part of large network of different pathways that intercept with each other, but these reactions don t occur at the same time if so they will reach end point and equilibrium. The metabolism is regulated so as the production of the metabolism pathways this consist with need of the cell, every cell has its own need, so metabolism should match the needs of the cell. However, in multi cellular organism cell do not function in isolation, there is sort of communication between the cells, as the function or activity of the cell reach the required needs of the organism. In order to coordinate between cells, metabolic pathway is controlled by: i. Intracellular Signals: arise in the cell itself. These signals typically rapid responses, and are short in duration. ii. Extracellular signals: (Communication between cells) usually results in a response that is slow and longer in duration. To coordinate the function of different cell for the greater for whole organism However, chemical signals that coordinate the cells function is hormones or neurotransmitter. This chemical compound to affect any cell, the cell must have receptor. Receptors should have a shape that is complementary to the shape of the signal molecule, so it bind to the receptor specifically, so the chemical signal will not affect the cell unless the cells have receptor. Binding of the chemical signal to the receptor is not enough a signal must be transmitted to inside the cell by second messenger. The second messenger carries the message to various enzymes which together made the cellular response to the chemical signal. 2

3 Receptors Many receptors are part of the plasma membrane of cells, and a common structure of receptors is seven transmembrane helices, the extracellular domain recognizes the shape of the signal molecule and the intracellular domain transmit a signal inside the cell (in this situation) by interacting with a protein in the plasma membrane called G-protein. G-protein consist of three sub unit (α, β, γ), when the receptor bound to hormone this will cause replacement of GDP with GTP, that will cause disassociation of the G- protein into 2 component α subunit and β, γ dimer, the α subunit that carry the GTP will react with other enzyme that found in the cell membrane called adenylyl cyclase that convert ATP into camp and PP i. camp will stimulate another protein called protein kinase that add phosphate groups to many enzymes, and adding the phosphate group to those enzymes will inhibit some of them and activate others, producing the cellular response. Glycolysis (Glyco: sugar, lysis: cleavage) In this process glucose will lysis into 2 pyruvate, and it s a universal pathway all cell have glycolysis and it done by the same manner. It is the first pathway to be discovered. It take place in the cytoplasm and it purpose is the production of energy in the form of ATP and production of building blocks which used in biosynthesis of other molecule, pyruvate for example is used in other pathway to produce amino acid, fatty acid, glycerol. Etc. So it has dual action (energy production, building blocks). It have 10 steps, so to understand it well, know the type of the reaction, then you will understand what happen, and you can name the enzyme. 3

4 Types of reaction: transfer of phosphate group, isomerization, cleavage, oxidation reduction reaction, dehydration. You must know these structure: glucose, fructose in the open chain formula, those two are hexoses and they are very similar, but glucose is aldose and fructose is ketose. Glyceraldehydes: derivative of glycerol (3 carbon with 3 OH group), if one carbon is oxidized to aldehyde it called glyceraldehydes, if this glyceraldehydes is oxidized to carboxylic acid it called glycerate. Glycolysis can be divided into 3 stages: Stage 1 : glucose is phosphorylated, and converted to Fructose, and phosphorylated again this occur by 3 steps. Stage 2: cleavage of the fructose 1,6-phosphate into 2 halves [ glyceraldehydes 3- phosphate(gap), dihydroxyacetone phosphate(dhap) ], each one have 3 C atoms and it s phosphorylated, they are aldotriose and ketotriose. Stage 3: oxidation of glyceraldehydes 3-phosphate, and addition of phosphate, then transfer of 1 P i to ADP to make ATP, then three more step we will have phosphoenolpyruvate, which can be conducted to pyruvate. This stage will produce 2 NADH per 1 GAP, So it produce 4 NADH per one glucose molecule. Only glyceraldehydes 3-phosphate can be taken directly, but it is in equilibrium with DHAP, and there is enzyme that converts DHAP to GAP and vice versa. 4

5 Glycolysis in Details STAGE 1: PHOSPHORYLATION OF GLUCOSE You have to add phosphate group, but we can t add it as such, it can be added only if the donar is ATP because hydrolysis of ATP produce 7.3 Cal, and synthesis of glucose 6-phosphate need only 3 Cal, so ATP molecule lose P i to glucose at carbon number 6 forming ester bond (between glucose and P i ) and ADP is produced. When P i is added to glucose it can t penetrate the membrane (has nevative charge) so it s kept (trapped) in the cell. Irreversible reaction: because ΔG for this reaction is (highly exergonic). The enzyme that catalyze this reaction is hexokinase, and because it is irreversible reaction it should be controlled, it is controlled by product, accumulation of G6P will inhibit the enzyme this called product inhibition, it is important step because if there is no control then there will be depletion of phosphate, if that happened there will be no ATP. It has broad specificity (it s obvious from the enzyme hexokinas). Hexo: 6 carbon, kinase: any enzyme that catalyse the transfer of P i from ATP to the substrate. Low K m : the K m for the enzyme is 1 mm, whereas the concentration of the glucose in the blood is 5 mm, that s mean it can utilize glucose even in low concentration, Like in hypoglycemia where Glucose concentration decreased for example from 5 mm to 3 mm but hexokinas still can function properly. K m : is the substrate concentration when the enzme velocity is 50% of the maximum velocity. 5

6 It appears like combination of 2 reactions: ATP is hydrolyzed and the phosphate is transferred. But in reality if that happened, the reaction will not occur. Because once the phosphate is hydrolyzed, it requires lot of energy to add it. What is good about this enzyme that it cannot allow water to replace glucose. So let s say that there was water here instead of glucose, the enzyme normally requires ATP but instead of binding to glucose (bind water) what will happen? Hydrolysis of ATP, but we said that hydrolysis of ATP will not happen because the enzyme excludes water and allows only glucose. This is how Hexokinase transfer phosphate only to glucose. Scientists studied the three dimensional structure of glucose. Why is there blue and red? Hexokinase in the absence of glucose is the blue one, and in the presence of glucose is the red one. When glucose binds it will cover it and the only remaining uncover part of glucose is the part on the surface where CH 2 OH is located the C number 6. ATP can t bind unless glucose is bound so there is no way for the ATP to be hydrolyzed and the formational changes that happen by binding of glucose will allow the ATP to bind, and now ATP hydrolysis is not in consideration. So now what is happening that water cannot be bound, excluded so there is no place for water, but there is place for ATP and just when the proper orientation it will be attacked by the OH group of glucose which is on the surface and it will attack the phosphate bond and form the Glucose-6-phosphate. Now, glucose-6-phosphate has many phases, not only the glycolysis, it s also used in the liver in glycogen synthesis and in other cells for producing other sugars like ribose and pentose, and it s used in producing NADPH which is similar to NADH except that it s used for reduction. So glucose-6-phosphate is the first step in utilization of glucose regardless what we are going to do next, that s why it s not strictly regulated because there are several pathways of using it. glycogenolysis happens in the liver where Glycolysis happens everywhere and it very important in cells, like in brain and RBC s because it s the only way and there is no way for using fatty acids, on the other side liver can use fatty acids and glucose as a source of energy. 6

7 When the liver start to make Glycogen? In the postabsorbic state,when you take food which contain carbohydrates, the glucose in the blood will start to elevate and then the synthesis of glycogen will take place. That s why in the liver there is another enzyme called Glucokinase, it s different form of the same enzyme and can work with other sugars than glucose and Glucokinase has high K m toward glucose, while the Hexokinase where is located in the muscles or the RBC s or the brain has low K m, so based on that the higher priority to use glucose goes for the organ that has Hexokinases with K m ; Brain, muscles and RBC s. The reasons for giving the highest priority for Hexokinases to use the Glucose; are: 1. Hexokinases can work with very low concentration of glucose because it have low K m. 2. Glycogen synthesis occurs when high concentration of glucose is available because Glucokinase has high K m. Two different forms of the same enzyme affect the biological function of two different tissues. Glucokinase can also called Hexokinase; because it doesn t only work on Glucose, but also can works on different sugars as well. ISOMERIZATION Isomerization of (G6P) converts it from Aldose into Ketose. During the Isomerization we wouldn t have to change all of carbon atoms, only carbon-1 and carbon-2. This change is huge; because we change the double bond site from carbon-1, in (G6P), to carbon-2, in (F6P). If we hide carbon-1 and carbon-2, we will not be able to distinguish between the Fructose and Glucose, while carbon-3, 4, 5 and 6 have the same orientation of the hydroxyl groups. 7

8 Carbon-1 in (G6P) has an Aldehyde group at carbon-1 and Hydroxyl (-OH) group at carbon-2. In contrast, (F6P) has a Hydroxyl (-OH) group at carbon-1 and Ketone group at carbon-2. The rest of the molecule is the same. The ring structure is more stable hence it s the predominant one. This event happens by Adding a Hydrogen at carbon-1 in (G6P) and removing a Hydrogen from carbon-2 and by that we transfer the Double bond from carbon-1 in (G6P) to carbon-2. So the (G6P) become (F6P). The isomerization is very easy and can be done without enzyme, by adding the Fructose in an Alkaline water (Little bit basic), and after a while we will have an equilibrium state between the Glucose and Fructose. This is called a NON- ENZYMATIC reaction, but would proceed in a very slow rate, and in order to increase the rate of this reaction, we have to use an enzyme called: PhosphoGlucose Isomerase. After the isomerization has been done, we will transfer the (F6P) from the open chain form into the closed one, which is the RING Structure ( 5-membered ring ). The isomerization reaction is a reversible reaction which means whatever was the reactant, we will end up with equilibrium between these both molecules. You should know that the reaction that converts the (F6P) into (G6P) proceeds more rapid than the reversible one. 8

9 ADDITION OF PHOSPHATE GROUP The next step that is going to occur is the addition of phosphate group on C 1 (the donor of the phosphate group is ATP) and we will get fructose 1,6-bisphosphate (on C1 and C6). We call it bisphosphate and not diphosphate because the 2 phosphates are not attached together as in ADP. The enzyme used here is phosphofructokinase. This step is a committed step which means that after this step we can t proceed anywhere but to glycolysis. Glucose-6-phosphate had more than one fate but fructose 1,6-bisphosphate is used only in glycolysis (after the addition of phosphate group you commit this sugar to glycolysis), so it should be strictly regulated and if u want to stop glycolysis you should stop this reaction not the first reaction because the first reaction will stop everything. We will talk more about regulation next lecture. STAGE 2: CLEAVAGE OF FRUCTOSE 1,6-BISPHOSPHATE Now is the cleavage of fructose 1, 6-bisphosphate. The cleavage happens between C3 and C4 and this is called aldol cleavage which is the opposite on aldol condensation and the enzyme here is Aldolase. This reaction gives 2 compounds : 1. glyceraldehyde 3-phosphate (similar to glycerol but with aldehyde group on C1 instead of alcohol and with phosphate on C3) 2. Dihydroxyacetone phosphate. 9

10 We can illustrate the characteristic of this reaction by this: The reaction is reversible and it s a cleavage reaction. The enzyme s class is lyase (cleaves the c-c bond without addition of water). On the site of cleavage there will be a double bond between C and O. ISOMERIZATION OF DIHYDROXYACETONE PHOSPHATE Because we need only glyceraldehydes 3-phosphate, the dihydroxyacetone phosphate will isomerizes into glyceraldehydes 3-phosphate and the enzyme for the reaction is isomerase (it s similar to the isomerization we discussed before; aldehyde to ketone), but here we convert from ketone to aldehyde. it s a reversible reaction. The number of atoms is the same. dehydrogenase always remove hydrogen in oxidation-reduction reaction and give it to FAD or NAD. This reaction can occur without the enzyme isomerase but it would be much slower but with isomerase enzyme it would be times faster. However, the only limiting factor for the reaction is the substrate finding the enzyme, if the substrate finds the enzyme, in no time the isomeration will occur. We should mention that Isomerase is very efficient (active) enzyme. 10

11 STAGE 3: Oxidation of glyceraldehyde 3-phosphate Glyceraldehyde 3-phosphate will be oxidized by glyceraldehyde 3-phosphate dehydrogenas, the aldehyde group will be oxidized to carboxyl group and the aldehyde will become carboxylic acid. Glyceraldehyde will become glycerate (acid) and the acid will immediately accept phosphate group to become acyle phosphate. The phosphate is linked to the carboxyl group by anhydride bond Now we can see that glyceraldehydes is oxidized and phosphorylated so it become 1,3-bisphosphoglycerate. Untile now we used 2 ATP and now it s time to harvest the ATP. Special thanks to: My Brother Omar, Amjad Alsharabati, M3tasim Al-Sayyed For helping with this sheet Sorry for any mistake Done By: Ammar AL-Habahbeh 11